Methods to increase transgene expression from bacterial-based delivery systems by co-expressing suppressors of the eukaryotic type I interferon response

ABSTRACT

Bacterial delivery systems with improved transgene expression are provided. The recombinant bacterial delivery systems deliver transgenes of interest and suppressors of the eukaryotic Type I interferon response to eukaryotic cells. Suppression of the eukaryotic Type I interferon response allows improved expression of the encoded transgene.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to bacterial delivery systems thatpromote improved transgene expression in eukaryotic cells by inhibitingthe innate type I interferon response. In particular, the inventionprovides recombinant bacterial delivery systems that deliver toeukaryotic cells: i) transgenes and ii) suppressors of the eukaryoticType I interferon response.

2. Background of the Invention

Live attenuated mutants of several pathogenic bacteria have beenexploited as potential vaccine vectors for heterologous antigen deliveryby the mucosal route. Such live vectors offer the advantage of targeteddelivery of macromolecules to mammalian cells and tissues in a singleoral, intranasal or inhalational dose, thereby stimulating both systemicand mucosal immune responses. The great potential of bacteria-mediatedtransfer of plasmid DNA encoding vaccine antigens and/or therapeuticmolecules has been demonstrated in experimental animal models ofinfectious diseases, tumors and gene deficiencies.

Unfortunately, bacterial vectored discharge of passenger RNA/DNA andother molecules for the expression of foreign proteins or inhibitoryRNAs in mammalian cells results in a type I interferon (IFN) response. Acentral component of the host's surveillance system for invadingpathogens is an evolutionarily conserved family of pathogen recognitionreceptors (PRR) which bind patterned microbial/viral ligands rangingfrom cell wall components to nucleic acids. PRR signaling results in theactivation of transcription factors such as Nuclear Factor-B (NF-B) andinterferon regulatory factor 3 (IRF-3), which provide the inflammatorycontext for the rapid activation of host defenses. The NF-B pathwaycontrols the expression of proinflammatory cytokines such as IL-1 andtumor necrosis factor-α, whereas the IRF-3 pathway leads to theproduction of type I interferons (IFN-α and IFN-β). This initiallyproduced “first wave” IFN triggers expression of a related factor,IRF-7, which is normally present in most cells at very lowconcentrations (Sato M et al., Immunity, 13(4)539-548; 2000). IRF-3 mostlikely cooperates with IRF-7 and is responsible for a positive feed backloop that initiates the synthesis of several IFN-α subtypes as the“second wave” IFNs (Marie et al., EMBO J. 17(22), 6660-6669; 1998 andSato M et al., FEBS Lett 441(1)106-110; 1998.). Type I IFNs activateseveral hundred IFN stimulated genes by autocrine and paracrinesignaling (ISGs) (de Veer et al., J Leukocyte Biol 69(6) 912-920, 2001;Der et al., Proc. Natl. Acad. Sci. USA 95(26) 15623-15628; 1998), someof which code for antiviral proteins. To date, three IFN stimulatedpathways have been firmly established. These include protein kinase R(PKR) (Williams Oncogene 18(45) 6112-6120; 1999), the 2′-5′oligoadenylate-synthetase (2′-5′ OAS) (Silverman., J Interferon Res14(3) 101-104; 1994) and the Mx proteins (Haller and Kochs Traffic 3(10)710-714; 2002). This type I IFN response limits the expression offoreign genes or inhibitory RNAs by means of PKR and 2′-5′ OAS.Activated PKR blocks translation by phosphorylating the a subunit ofeukaryotic initiation factor eIF2. On the other hand, 2-5A synthetasesproduce short, 2′-5′ OAS associated oligoadenylates which activate RNaseL, a single-stranded specific endoribonuclease that digests mRNA andribosomal RNA. The importance of the Mx protein in host survivalfollowing infection with certain RNA viruses has been amply demonstrated(Hefti et al., J Virology 73(8) 6984-6991; 1999) but the exact mode ofaction is still unknown. This type I IFN response thus limits theexpression of foreign nucleic acids by mechanisms which reduce RNAproduction and stability and also inhibits translation of message frompassenger nucleic acids delivered by a bacterial vector.

Various components of bacterial vectors elicit the IFN response in hostcells. The bacterium itself can trigger an IFN response throughToll-like receptors. Double stranded RNA produced by passenger nucleicacids during transcription not only induces type I IFNs but alsodirectly activates PKR and 2′-5′ OAS. Plasmid DNA, upon its deliveryinto the cytoplasm of mammalian cells, often contains cryptic promotersthat generate anti-sense RNA which anneals with mRNA to form dsRNA. Allthese components of bacterial vectors thus diminish the efficacy ofbacterial vectors as biomedical tools.

U.S. Pat. No. 6,525,029 (Falck-Perersen et al., Feb. 25, 2003) describesmethods of inhibiting an immune response to a recombinant vector such asan adenoviral vector. However, this technology is directed towardpreventing humoral (e.g. antibody) responses to long-term expression ofgenes encoded by a vector and clearance of the vector by the immunesystem, and does not address prevention of a type I IFN response to abacterial vector or its passenger nucleic acids.

The prior art has thus-far failed to provide bacterial vectors thateliminate or attenuate the type I IFN response of host cells.

SUMMARY OF THE INVENTION

The present invention provides recombinant bacterial expression vectorsthat successfully eliminate or attenuate the type I IFN response that isusually mounted by mammalian host cells in response to invasion by abacterial expression vector. The recombinant bacterial expressionvectors circumvent the usual IFN response by encoding factors thatinhibit or suppress the type I IFN response in host cells. The IFNsuppressor is expressed either i) in the bacterial cell for delivery asa protein or ii) in the eukaryotic cell from a nucleotide sequence thatis delivered by the bacterial cell. Inhibition of the IFN responseallows more robust expression of passenger genes delivered by thebacterial vector, and expression is enhanced only in a eukaryotic cellin which the type I IFN response has been suppressed. For example, whenthe recombinant bacterial expression vector of the invention deliverspassenger nucleotide sequences encoding antigens to which an immuneresponse is desired, production of those antigens by the mammalian cellis not impeded by the host IFN system, the antigens are expressed, andthe desired immune response to the antigens may be produced.

It is an object of this invention to provide a genetically engineeredbacterium, comprising nucleic acid sequences encoding i) one or morepassenger genes; and ii) one or more factors that inhibit a mammalianinterferon response. The nucleic acid sequences encoding the one or morepassenger genes are operably linked to a eukaryotic promoter, and thenucleic acid sequences encoding the one or more factors that inhibit amammalian type I interferon response are operably linked to a eukaryoticpromoter or a prokaryotic promoter. In yet another embodiment, theexpressible nucleic acid sequences encoding the one or more factors thatinhibit a mammalian interferon response are present on a chromosome ofthe genetically engineered bacterium. In further embodiments, one orboth of the: i) nucleic acid sequences encoding said one or morepassenger genes, wherein the nucleic acid sequences are expressible in aeukaryotic cell; and ii) nucleic acid sequences encoding said one ormore factors that inhibit a mammalian interferon response, are presenton a plasmid. In addition, the one or more factors that inhibit amammalian interferon response may be of viral origin. In someembodiments, the one or more passenger genes encode tuberculosisantigens. In further embodiments, the genetically engineered bacteriumis a shigella bacterium or a mycobacterium. Further, the passenger genesmay be heterologous transgenes.

The invention further provides a method of increasing the production ofone or more gene products of interest in a cell or tissue. The methodcomprises the step of administering to the cell or tissue a geneticallyengineered bacterium comprising nucleic acid sequences encoding: i) theone or more gene products of interest and ii) one or more factors thatinhibit a mammalian interferon response. The nucleic acid sequencesencoding the one or more passenger genes are operably linked to aeukaryotic promoter, and the nucleic acid sequences encoding the one ormore factors that inhibit a mammalian type I interferon response areoperably linked to a eukaryotic promoter or a prokaryotic promoter. Thestep of administering is carried out under conditions which allow thegenetically engineered bacterium to invade the cell or tissue, and toproduce the one or more gene products of interest and the one or morefactors within the cell or tissue. In one embodiment, transcription ofthe expressible nucleic acid sequences is controlled by eukaryoticpromoters. In another embodiment, transcription of the expressiblenucleic acid sequences encoding the one or more factors that inhibit amammalian interferon response is controlled by prokaryotic promoters. Inyet another embodiment, the expressible nucleic acid sequences encodingthe one or more factors that inhibit a mammalian interferon response arepresent on a chromosome of the genetically engineered bacterium. Infurther embodiments, one or both of: i) expressible nucleic acidsequences encoding the one or more gene products of interest, and ii)expressible nucleic acid sequences encoding the one or more factors thatinhibit a mammalian interferon response, are present on a plasmid. Inaddition, the one or more factors that inhibit a mammalian interferonresponse may be of viral origin. In some embodiments, the one or moregene products of interest may be tuberculosis antigens. In furtherembodiments, the genetically engineered bacterium is a shigellabacterium or a mycobacterium.

The invention further provides a method for inducing an immune responseto an antigen of interest in a mammal. The method comprises the step ofadministering to the mammal a genetically engineered bacterium,comprising nucleic acid sequences encoding the antigen of interest; andnucleic acid sequences encoding one or more factors that inhibit amammalian interferon response. The nucleic acid sequences encoding theone or more passenger genes are operably linked to a eukaryoticpromoter, and the nucleic acid sequences encoding the one or morefactors that inhibit a mammalian type I interferon response are operablylinked to a eukaryotic promoter or a prokaryotic promoter. In oneembodiment of the invention, the antigen of interest is a Mycobacteriumtuberculosis antigen. In some embodiments, transcription of theexpressible nucleic acid sequences is controlled by eukaryoticpromoters. In other embodiments, transcription of the expressiblenucleic acid sequences encoding the one or more factors that inhibit amammalian interferon response is controlled by prokaryotic promoters. Inyet other embodiments, the expressible nucleic acid sequences encodingthe one or more factors that inhibit a mammalian interferon response arepresent on a chromosome of the genetically engineered bacterium. In someembodiments, one or both of: i) expressible nucleic acid sequencesencoding the antigen of interest, and ii) expressible nucleic acidsequences encoding the one or more factors that inhibit a mammalianinterferon response, are present on a plasmid. In further embodiments,the one or more factors that inhibit a mammalian interferon response areof viral origin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Beta-galactosidase activity of cell lysates after invasion ofHeLa or BHK-21 cells (IFN deficient) with Shigella flexneri NCD1carrying a plasmid encoding eukaryotic expression of β-galactosidase.Black bar indicates β-galactosidase activity from cells post invasionwith a bacterial strain harboring a plasmid encoding the lacZ gene;white bar indicates β-galactosidase activity from cells post invasionwith a bacterial strain minus the lacZ plasmid.

FIG. 2. β-galactosidase activity of lysates of HeLa cells after invasionwith Shigella flexneri NCD1 harboring a plasmid encoding β-galactosidaseand after co-invasion with Shigella flexneri NCD1 encoding an adenovirusderived inhibitor of PKR (adenovirus-associated I, VAI).

FIG. 3. Immunoblot showing transgene expression of green fluorescentprotein (GFP) protein in HeLa cells post invasion with Shigella flexneristrain MPC51 which carries a eukaryotic GFP reporter gene only (lane 4)or GFP plus NS1(lane 5) or NSP1 (lane 2). Lane 1: positive control; Lane3: non-invaded HeLa cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The recombinant bacterial expression vectors of the present inventionare genetically engineered to encode factors that eliminate, attenuateor suppress the type I interferon response that is usually mounted bymammalian host cells in response to invasion by a bacterium. Thesefactors may be expressed by the bacterial vector cell or may be encodedin nucleic acids which are translated in the eukaryotic host cell.Attenuation or elimination of the IFN response in the eukaryotic hostcell permits efficient transcription and translation of proteins andpeptides of interest from vector introduced nucleic acids. Such vectoredmolecules may encode peptides and proteins that are necessary for thebacteria's reproduction and survival, as well as “passenger” moleculesof interest contained within the bacterium. Examples of passengernucleic acids of interest include but are not limited to, for example,antigens that the bacterium has been genetically engineered to encode.Because the eukaryotic host cell's type I IFN response is attenuated,the antigens are expressed persistently and at a level sufficient tocause the host cell to mount an immune response to the antigens. Thebacterial expression vectors of the invention are thus ideal for use invaccine preparations.

The bacterial expression vectors of the invention are geneticallyengineered to encode factors that eliminate, attenuate or suppress thetype I IFN response. Those of skill in the art will recognize that manyviruses encode factors that target specific mediators of IFN responses.These factors can be referred to as IFN response antagonists. Among thebest characterized viral targets are protein kinase R (PKR), RNaseLactivating (2′-5′) oligoadenylate synthetase and the InterferonRegulatory Factor (IRF) family of proteins.

Such mechanisms are encoded by a variety of viruses, examples of whichinclude but are not limited to: rotavirus non structural protein1(NSP1); influenza-A virus non structural protein 1 (NS1); adenovirusassociated RNA I and II (VAI and II); vaccinia virus E3L; hepatitis Cvirus non structural protein 5A (NS5A); simian virus-V protein; Sendaivirus C protein; etc.

While in some embodiments, the factors that inhibit the IFN response arederived from viruses, such factors may be obtained from other sources,for example, from the host cell (e.g. suppressors of cytokine signaling,SOCS), dominant negative alleles of PKR and dominant negative alleles ofRNaseL) and may be utilized in the practice of the present invention.Any factor that suppresses or attenuates the type I IFN response andwhich is encoded by a nucleic acid sequence that can be geneticallyengineered into and successfully expressed from a bacterial expressionvector or delivered to eukaryotic cells by a bacterial vector may beused in the practice of the present invention.

By “bacterial expression vector” we mean a bacterial cell that has beengenetically engineered to contain and express or deliver nucleic acidsequences of interest. Examples of bacteria which can be utilized inthis manner include but are not limited to Campylobacter spp, Neisseriaspp., Haemophilus spp, Aeromonas spp, Francisella spp, Yersinia spp,Klebsiella spp, Bordetella spp, Legionella spp, Corynebacterium spp,Citrobacter spp, Chlamydia spp, Brucella spp, Pseudomonas spp,Helicobacter spp, or Vibrio spp.

The particular Campylobacter strain employed is not critical to thepresent invention. Examples of Campylobacter strains that can beemployed in the present invention include but are not limited to: C.jejuni (ATCC Nos. 43436, 43437, 43438), C. hyointestinalis (ATCC No.35217), C. fetus (ATCC No. 19438) C. fetalis (ATCC No. 33709) C. doylei(ATCC No. 49349) and C. coli (ATCC Nos. 33559, 43133).

The particular Yersinia strain employed is not critical to the presentinvention. Examples of Yersinia strains which can be employed in thepresent invention include: Y. enterocolitica (ATCC No. 9610) or Y.pestis (ATCC No. 19428), Y. enterocolitica Ye03-R2 (al Hendy et al.,Infect. Immun., 60:870; 1992) or Y. enterocolitica aroA (O'Gaora et al.,Micro. Path., 9:105; 1990).

The particular Klebsiella strain employed is not critical to the presentinvention. Examples of Klebsiella strains that can be employed in thepresent invention include K. pneumoniae (ATCC No. 13884).

The particular Bordetella strain employed is not critical to the presentinvention. Examples of Bordetella strains which can be employed in thepresent invention include B. pertussis, and B. bronchiseptica (ATCC No.19395).

The particular Neisseria strain employed is not critical to the presentinvention. Examples of Neisseria strains that can be employed in thepresent invention include N. meningitidis (ATCC No. 13077) and N.gonorrhoeae (ATCC No. 19424), N. gonorrhoeae MS11 aro mutant(Chamberlain et al., Micro. Path., 15:51-63; 1993).

The particular Aeromonas strain employed is not critical to the presentinvention. Examples of Aeromonas strains that can be employed in thepresent invention include A. salminocida (ATCC No. 33658), A. schuberii(ATCC No. 43700), A. hydrophila, A. eucrenophila (ATCC No. 23309).

The particular Francisella strain employed is not critical to thepresent invention. Examples of Francisella strains that can be employedin the present invention include F. tularensis (ATCC No. 15482).

The particular Corynebacterium strain employed is not critical to thepresent invention. Examples of Corynebacterium strains that can beemployed in the present invention include C. pseudotuberculosis (ATCCNo. 19410).

The particular Citrobacter strain employed is not critical to thepresent invention. Examples of Citrobacter strains that can be employedin the present invention include C. freundii (ATCC No. 8090).

The particular Chlamydia strain employed is not critical to the presentinvention. Examples of Chlamydia strains that can be employed in thepresent invention include C. pneumoniae (ATCC No. VR1310).

The particular Haemophilus strain employed is not critical to thepresent invention. Examples of Haemophilus strains that can be employedin the present invention include H. influenzae (Lee et al., J. Biol.Chem. 270:27151; 1995), H. somnus (ATCC No. 43625).

The particular Brucella strain employed is not critical to the presentinvention. Examples of Brucella strains that can be employed in thepresent invention include B. abortus (ATCC No. 23448).

The particular Legionella strain employed is not critical to the presentinvention. Examples of Legionella strains that can be employed in thepresent invention include L. pneumophila (ATCC No. 33156), or a L.pneumophila mip mutant (Ott, FEMS Micro. Rev., 14:161; 1994).

The particular Pseudomonas strain employed is not critical to thepresent invention. Examples of Pseudomonas strains that can be employedin the present invention include P. aeruginosa (ATCC No. 23267).

The particular Helicobacter strain employed is not critical to thepresent invention. Examples of Helicobacter strains that can be employedin the present invention include H. pylori (ATCC No. 43504), H. mustelae(ATCC No. 43772).

The particular Vibrio strain employed is not critical to the presentinvention. Examples of Vibrio strains that can be employed in thepresent invention include Vibrio cholerae (ATCC No. 14035), Vibriocincinnatiensis (ATCC No. 35912), V. cholerae RSI virulence mutant(Taylor et al., J. Infect. Dis., 170:1518-1523; 1994) and V. choleraectxA, ace, zot, cep mutant (Waldor J et al., Infect. Dis., 170:278-283;1994).

In a preferred embodiment, the bacterial strain from which the vectorstrain is developed in the present invention includes bacteria thatpossess the potential to serve both as a carrier and as a vaccinevectors, such as the Enterobacteriaceae, including but not limited toEscherichia spp, Shigella spp, and Salmonella spp. Gram-positive andacid-fast vector strains could similarly be constructed from Listeriamonocytogenes or Mycobacterium spp.

The particular Escherichia strain employed is not critical to thepresent invention. Examples of Escherichia strains which can be employedin the present invention include Escherichia coli strains DH5α, HB 101,HS-4, 4608-58, 1184-68, 53638-C-17, 13-80, and 6-81 (See, e.g. Sambrooket al., supra; Grant et al., supra; Sansonetti et al., Ann. Microbiol.(Inst. Pasteur), 132A:351; 1982), enterotoxigenic E. coli (See, e.g.Evans et al., Infect. Immun., 12:656; 1975), enteropathogenic E. coli(See, e.g. Donnenberg et al., J. Infect. Dis., 169:831; 1994),enteroinvasive E. coli (See, e.g. Small et al., Infect Immun., 55:1674;1987) and enterohemorrhagic E. coli (See, e.g. McKee and O'Brien,Infect. Immun., 63:2070; 1995).

The particular Salmonella strain employed is not critical to the presentinvention. Examples of Salmonella strains that can be employed in thepresent invention include S. typhi (see, e.g. ATCC No. 7251), S.typhimurium (see, e.g. ATCC No. 13311), Salmonella galinarum (ATCC No.9184), Salmonella enteriditis (see, e.g. ATCC No. 4931) and Salmonellatyphimurium (see, e.g. ATCC No. 6994). S. typhi aroC, aroD double mutant(see, e.g. Hone et al., Vacc., 9:810-816; 1991), S. typhimurium aroAmutant (see, e.g. Mastroeni et al., Micro. Pathol., 13:477-491; 1992).

The particular Shigella strain employed is not critical to the presentinvention. Examples of Shigella strains that can be employed in thepresent invention include Shigella flexneri (see, e.g. ATCC No. 29903),Shigella flexneri CVD1203 (see, e.g. Noriega et al., Infect. Immun.62:5168; 1994), Shigella flexneri 15D (see, e.g. Sizemore et al.,Science 270:299; 1995), Shigella sonnei (see, e.g. ATCC No. 29930), andShigella dysenteriae (see, e.g. ATCC No. 13313).

The particular Mycobacterium strain employed is not critical to thepresent invention. Examples of Mycobacterium strains that can beemployed in the present invention include M. tuberculosis CDC1551 strain(See, e.g. Griffith et al., Am. J. Respir. Crit. Care Med. August;152(2):808; 1995), M. tuberculosis Beijing strain (Soolingen et al.,1995) H37Rv strain (ATCC#:25618), M. tuberculosis pantothenate auxotrophstrain (Sambandamurthy, Nat. Med. 2002 8(10):1171; 2002), M.tuberculosis rpoV mutant strain (Collins et al., Proc Natl Acad Sci USA.92(17):8036; 1995), M. tuberculosis leucine auxotroph strain (Hondaluset al., Infect. Immun. 68(5):2888; 2000), Bacille Calmette-Guérin (BCG)Danish strain (ATCC #35733), BCG Japanese strain (ATCC #35737), BCG,Chicago strain (ATCC #27289), BCG Copenhagen strain (ATCC #: 27290), BCGPasteur strain (ATCC #: 35734), BCG Glaxo strain (ATCC #: 35741), BCGConnaught strain (ATCC #35745), BCG Montreal (ATCC #35746).

The particular Listeria monocytogenes strain employed is not critical tothe present invention. Examples of Listeria monocytogenes strains whichcan be employed in the present invention include L. monocytogenes strain10403S (e.g. Stevens et al., J. Virol 78:8210-8218; 2004) or mutant L.monocytogenes strains such as (i) actA plcB double mutant (Peters etal., FEMS Immunology and Medical Microbiology 35: 243-253; 2003);(Angelakopoulous et al., Infect and Immunity 70: 3592-3601; 2002); (ii)dal dat double mutant for alanine racemase gene and D-amino acidaminotransferase gene (Thompson et al., Infect and Immunity66:3552-3561; 1998).

In some embodiments of the invention, the bacteria are, in particular,Shigella species, in particular attenuated invasive Shigella flexneri2a. These strains, MPC51 and NCD1 are derivatives of S. flexneri strain2457T into which asd and murI deletion mutations have been introduced.The asd defect is complemented by the expression vector encoded asdallele and the murI mutation results in the inability of the strain tosynthesize D-glutamate; hence, these strains are incapable ofsynthesizing a proper cell wall in the absence of diaminopimelic acidand D-glutamate, which promotes lysis of the bacterial cell afterinvasion of a eukaryotic cell. As measured by a gentamicin protectionassay, the HeLa cell invasive behavior of the Δasd, ΔmurI double mutantMPC51 was similar to that of the parental strain and MPC51pYA3342(plasmid encoding asd). The strain has been further modified by removalof the kanamycin resistance gene previously inserted in the chromosomalasd locus. The resultant strain, Shigella flexneri NCD1, is thus free ofantibiotic resistance markers, still retains chromosomal deletions ofthe asd and murI genes, and is acceptable for pharmacologic use inhumans under current regulatory requirements. NCD1 has also been shownto be invasive in HeLa and Caco-2 cells in a manner similar to theparent strain.

Generally, the bacterial expression vectors of the invention aregenetically engineered to encode and deliver both the IFN inhibitingfactors and one or more other genes of interest i.e. passenger genes.The passenger genes are typically heterologous transgenes that originatefrom another organism, such as another bacteria or pathogen, and may befrom any organism. However, the “passenger gene” may also be a gene thatnaturally occurs in the bacterial vector itself (i.e. is derived from ororiginates from the bacteria that serves as a vector), but one or moreadditional copies are genetically engineered in the bacterial vector tobe under the control of a promoter that, for example, increases thelevel of transcription above that which is typical for the bacteria, ora promoter that is specific for a particular type of host cell or tissue(e.g. lung, lymph node, dendritic cell, etc). Further, “passenger gene”is intended to refer not only to entire “genes” but to any sequence thatencodes a peptide, polypeptide, protein, or nucleic acid of interest,i.e. an entire “gene” per se may not be included, but rather the portionof a gene that encodes a polypeptide or peptide of interest e.g. anantigenic peptide. Further, various other constructions may be encodedby passenger genes, e.g. chimeric proteins, or various mutant (eithernaturally occurring or genetically engineered) forms of an amino acidsequence. In addition, totally artificial amino acid sequences that donot appear in nature may also be encoded. The bacterial expressionvector is genetically engineered to contain one or more of such“passenger genes”, and may also encode multiple copies of individualpassenger genes. The recombinant bacterial expression vector functionsas a vector to carry the passenger gene(s) into host cells that areinvaded by the bacterium, where the gene product is expressed, i.e. thegene sequences are expressible and transcription and/or translation ofthe gene product occurs within the host cell that is invaded by thebacterium. The sequences encoding the passenger genes are operatively(operably) linked to expression control sequences, particularlyexpression control sequences that allow expression within the eukaryotichost cell.

In particular, such passenger genes may encode one or more peptides orproteins that are antigens, and to which it is desired to elicit animmune response. Those of skill in the art will recognize that a widevariety of such antigens exists, including but not limited to thoseassociated with infectious agents such as various viruses, bacteria, andfungi, etc. The viral pathogens, from which the viral antigens arederived, include, but are not limited to, Orthomyxoviruses, such asinfluenza virus (Taxonomy ID: 59771; Retroviruses, such as RSV, HTLV-1(Taxonomy ID: 39015), and HTLV-II (Taxonomy ID: 11909), Papillomaviridaesuch as HPV (Taxonomy ID: 337043), Herpesviruses such as EBV TaxonomyID: 10295); CMV (Taxonomy ID: 10358) or herpes simplex virus (ATCC #:VR-1487); Lentiviruses, such as HIV-1 (Taxonomy ID: 12721) and HIV-2Taxonomy ID: 11709); Rhabdoviruses, such as rabies; Picornoviruses, suchas Poliovirus (Taxonomy ID: 12080); Poxviruses, such as vaccinia(Taxonomy ID: 10245); Rotavirus (Taxonomy ID: 10912); and Parvoviruses,such as adeno-associated virus 1 (Taxonomy ID: 85106).

Examples of viral antigens can be found in the group including but notlimited to the human immunodeficiency virus antigens Nef (NationalInstitute of Allergy and Infectious Disease HIV Repository Cat. #183;Genbank accession #AF238278), Gag, Env (National Institute of Allergyand Infectious Disease HIV Repository Cat. #2433; Genbank accession#U39362), Tat (National Institute of Allergy and Infectious Disease HIVRepository Cat. #827; Genbank accession #M13137), mutant derivatives ofTat, such as Tat-31-45 (Agwale et al., Proc. Natl. Acad. Sci. USA99:10037; 2002), Rev (National Institute of Allergy and InfectiousDisease HIV Repository Cat. #2088; Genbank accession #L14572), and Pol(National Institute of Allergy and Infectious Disease HIV RepositoryCat. #238; Genbank accession #AJ237568) and T and B cell epitopes ofgp120 (Hanke and McMichael, AIDS Immunol Lett., 66:177; 1999); (Hanke,et al., Vaccine, 17:589; 1999); (Palker et al., J. Immunol., 142:36123619; 1989) chimeric derivatives of HIV-1 Env and gp120, such as but notrestricted to fusion between gp120 and CD4 (Fouts et al., J. Virol.2000, 74:11427-11436; 2000); truncated or modified derivatives of HIV-1env, such as but not restricted to gp140 (Stamatos et al., J Virol,72:9656-9667; 1998) or derivatives of HIV-1 Env and/or gp140 thereof(Binley, et al., J Virol, 76:2606-2616; 2002); (Sanders, et al., JVirol, 74:5091-5100 (2000); (Binley, et al. J Virol, 74:627-643; 2000),the hepatitis B surface antigen (Genbank accession #AF043578); (Wu etal., Proc. Natl. Acad. Sci., USA, 86:4726 4730; 1989); rotavirusantigens, such as VP4 (Genbank accession #AJ293721); (Mackow et al.,Proc. Natl. Acad. Sci., USA, 87:518 522; 1990) and VP7 (GenBankaccession #AY003871); (Green et al., J. Virol., 62:1819 1823; 1988),influenza virus antigens such as hemagglutinin or (GenBank accession#AJ404627); (Pertmer and Robinson, Virology, 257:406; 1999);nucleoprotein (GenBank accession #AJ289872); (Lin et al., Proc. Natl.Acad. Sci., 97: 9654-9658; 2000) herpes simplex virus antigens such asthymidine kinase (Genbank accession #AB047378; (Whitley et al., In: NewGeneration Vaccines, pages 825-854).

The bacterial pathogens, from which the bacterial antigens are derived,include but are not limited to: Mycobacterium spp., Helicobacter pylori,Salmonella spp., Shigella spp., E. coli, Rickettsia spp., Listeria spp.,Legionella pneumoniae, Pseudomonas spp., Vibrio spp., Bacillus anthracisand Borellia burgdorferi.

Examples of protective antigens of bacterial pathogens include thesomatic antigens of enterotoxigenic E. coli, such as the CFA/I fimbrialantigen (Yamamoto et al., Infect. Immun., 50:925 928; 1985) and thenontoxic B subunit of the heat labile toxin (et al., Infect. Immun.,40:888-893; 1983); pertactin of Bordetella pertussis (Roberts et al.,Vacc., 10:43-48; 1992), adenylate cyclase hemolysin of B. pertussis(Guiso et al., Micro. Path., 11:423-431; 1991), fragment C of tetanustoxin of Clostridium tetani (Fairweather et al., Infect. Immun., 58:13231326; 1990), OspA of Borellia burgdorferi (Sikand et al., Pediatrics,108:123-128; 2001); (Wallich et al., Infect Immun, 69:2130-2136; 2001),protective paracrystalline-surface-layer proteins of Rickettsiaprowazekii and Rickettsia typhi (Carl et al., Proc Natl Acad Sci USA,87:8237-8241; 1990), the listeriolysin (also known as “Llo” and “Hly”)and/or the superoxide dismutase (also know as “SOD” and “p60”) ofListeria monocytogenes (Hess, J., et al., Infect. Immun. 65:1286-92;1997); Hess, J., et al., Proc. Natl. Acad. Sci. 93:1458-1463; 1996);(Bouwer et al., J. Exp. Med. 175:1467-71; 1992), the urease ofHelicobacter pylori (Gomez-Duarte et al., Vaccine 16, 460-71; 1998);(Corthesy-Theulaz, et al., Infection & Immunity 66, 581-6; 1998), andthe Bacillus anthracis protective antigen and lethal factorreceptor-binding domain (Price, et al., Infect. Immun. 69, 4509-4515;2001).

The parasitic pathogens, from which the parasitic antigens are derived,include but are not limited to: Plasmodium spp., such as Plasmodiumfalciparum (ATCC#: 30145); Trypanosome spp., such as Trypanosoma cruzi(ATCC#: 50797); Giardia spp., such as Giardia intestinalis (ATCC#:30888D); Boophilus spp., Babesia spp., such as Babesia microti (ATCC#:30221); Entamoeba spp., such as Entamoeba histolytica (ATCC#: 30015);Eimeria spp., such as Eimeria maxima (ATCC#40357); Leishmania spp.(Taxonomy ID: 38568); Schistosome spp., Brugia spp., Fascida spp.,Dirofilaria spp., Wuchereria spp., and Onchocerea spp.

Examples of protective antigens of parasitic pathogens include thecircumsporozoite antigens of Plasmodium spp. (Sadoff et al., Science,240:336 337; 1988), such as the circumsporozoite antigen of P. bergheior the circumsporozoite antigen of P. falciparum; the merozoite surfaceantigen of Plasmodium spp. (Spetzler et al., Int. J. Pept. Prot. Res.,43:351-358; 1994); the galactose specific lectin of Entamoebahistolytica (Mann et al., Proc. Natl. Acad. Sci., USA, 88:3248-3252;1991), gp63 of Leishmania spp. (Russell et al., J. Immunol., 140:12741278; 1988); (Xu and Liew, Immunol., 84: 173-176; 1995), gp46 ofLeishmania major (Handman et al., Vaccine, 18:3011-3017; 2000)paramyosin of Brugia malayi (Li et al., Mol. Biochem. Parasitol.,49:315-323; 1991), the triose-phosphate isomerase of Schistosoma mansoni(Shoemaker et al., Proc. Natl. Acad. Sci., USA, 89:1842 1846; 1992); thesecreted globin-like protein of Trichostrongylus colubriformis (Frenkelet al., Mol. Biochem. Parasitol., 50:27-36; 1992); theglutathione-S-transferase's of Frasciola hepatica (Hillyer et al., Exp.Parasitol., 75:176-186; 1992), Schistosoma bovis and S. japonicum(Bashir et al., Trop. Geog. Med., 46:255-258; 1994); and KLH ofSchistosoma bovis and S. japonicum (Bashir et al., supra, 1994).

Alternatively, it may be desired to elicit an immune response toantigens that are not associated with infectious agents, for example,antigens associated with cancer cells, Alzheimer's disease, Type 1diabetes, heart disease, Crohn's disease, multiple sclerosis, etc.

In addition, the passenger genes that are carried by the bacterium neednot encode antigens, but may encode any peptide or protein of interest.For example, the methods of the invention can be used for the deliveryof passenger molecules for correction of hereditary disorders. Suchgenes would include, for example, replacement of defective genes such asthe cystic fibrosis transmembrane conductance regulator (CFTR) gene forcystic fibrosis; or the introduction of new genes such as the integraseantisense gene for the treatment of HIV; or genes to enhance Type I Tcell responses such as interleukin-27 (IL-27); or genes to modulate theexpression of certain receptors, metabolites or hormones such ascholesterol and cholesterol receptors or insulin and insulin receptors;or genes encoding products that can kill cancer cells such as tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL); or anaturally occurring protein osteoprotegerin (OPG) that inhibits boneresorption; or to efficiently express complete-length humanizedantibodies, for example, humanized monoclonal antibody that acts on theHER2/neu (erbB2) receptor on cancer cells.

In addition, the passenger genes may encode inhibitory RNAs such as“small inhibitory” siRNAs. As is known in the art, such RNAs arecomplementary to an mRNA of interest and bind to and prevent translationof the mRNA, e.g. as a means of preventing the expression of a geneproduct.

Similar methods can be used for delivery of passenger molecules to downregulate the immune system in order to prevent or control autoimmunediseases or other diseases of immune system. Examples include theprevention or treatment of diabetes mellitus, multiple sclerosis, lupuserythematosis and Crohn's disease and inflammatory joint and skindiseases. Other examples include fine tuning of immune responses thathamper specific immune responses such as down regulation of immuneresponses that divert the therapeutic immune responses to cancer andother diseases. For example, down regulation of Th2 responses when Th1responses are appropriate for prevention and treatment of cancer,Leishmaniasis, tuberculosis, and HIV. This can be achieved by means ofthe present technology through manipulation of the immunosuppressivenature of the immune system in combination with the ability to expressthe suitable cytokine milieu for stimulation of the proper immuneresponse and inhibition of improper immune responses.

In a preferred embodiment, the present invention relates to a method forthe introduction of IFN resistance genes into host cells. Such a methodwould comprise introduction of the desired IFN resistance genes, alongwith sequences encoding a gene or nucleic acid sequence of interest,into a bacterial based delivery system such that the IFN resistanceproteins and nucleic acid sequences of interest are expressed uponadministering the bacteria to a host. The IFN inhibitor can be producedby the bacteria (e.g. shigella) or by the host cell. In other words, theIFN resistance genes can be expressed from a prokaryotic promoter orfrom a eukaryotic promoter. The gene or nucleic acid sequences ofinterest (passenger genes) are expressed by the host. Further, allgenetic sequences may be either constitutively expressed or induced.

In yet another preferred embodiment, the present invention provides amethod for the introduction of type I IFN resistance genes along withone or more genes of interest into cells in vitro. Such a method wouldcomprise introduction of the genes encoding one or more proteins ofinterest along with one or more IFN resistance genes into, for example,attenuated or attenuated/inactivated shigella such that the desiredproteins/peptides are produced upon administering the shigella to cells.Shigella infects several different cell types, such as BHK (baby hamsterkidney cells), HeLa (human cervical epitheloid carcinoma), CaCo-2 (humancolonic adenocarcinoma) and therefore is capable of delivering thedesired passenger molecules into cells. Gene expression in theshigella-infected cells is enhanced by the inhibitor of the type I IFNresponse. Following nucleic acid delivery, the cells can be transplantedfor therapeutic purposes, for gene therapy or used as reagents indiagnostic assays.

In these cases, the bacteria serve as “gene therapy” agents bydelivering to the cell nucleic acid sequences that encode a desiredsubstance and mediating its production in the cell. For example,delivering CXCR4/or CCR5 binding chemokine-encoding genes into the gutusing shigella vectors could be considered for treatment for HIV-1infection. Procedures for genetically engineering bacteria arewell-known to those of skill in the art, and guidance for carrying outsuch procedures are well known. Methods to attenuate E. coli,Salmonella, Mycobacteria, Shigella, and Listeria are well known to thoseskilled in the art (Evans et al., J. of Immuno., vol. 120, 1978, p.1423); (Noriega et al., Infect. Immun., 62(11):5168-5172 1994); (Hone etal., Vacc., 9:810-816; 1991).

For example, a method for the delivery of a desired gene or genes into acell may include introducing the gene of interest into a strain ofbacteria. In accordance with the present invention, an anti-IFN responsegene or genes can be introduced into the bacterial chromosome orvirulence plasmid by methods well known to those of skill in the art oralternatively can be carried in a replicating or nonreplicating plasmid.The vectors of interest can be introduced into the bacterium, forexample, via transformation, electroporation, transfection, conjugation,etc. The recombinant DNA procedures used in the construction of thestrains and bacterial vectors include but are not limited to: polymerasechain reaction (PCR), restriction endonuclease (herein referred to as“RE”) digestions, DNA ligation, agarose gel electrophoresis, DNApurification, and dideoxynucleotide sequencing, which are describedelsewhere (Miller, A Short Course in Bacterial Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; 1992); (Bothwell etal., Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jonesand Bartlett Publishers Inc., Boston, Mass. 1990); and (Ausubel et al.,Current Protocols in Molecular Biology, vol. 2:10.8.1-10.8.13, 1992),bacteriophage-mediated transduction (de Boer et al., Cell, 56:641-649;1989); (Miller, supra, 1992) and (Ausubel et al., supra), or chemical(Bothwell et al., supra); (Ausubel et al., supra); (Feigner et al.,supra); and (Farhood, supra), electroporation (Bothwell et al., supra);(Ausubel et al., supra); (Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;1992) and physical transformation techniques (Bothwell et al., supra).The genes can be incorporated in phage (de Boer et al., supra), plasmidsvectors (Curtiss, In: New Generation Vaccines: The Molecular Approach,Ed., Marcel Dekker, Inc., New York, N.Y., pages 161-188 and 269-2881989) or spliced into the chromosome (Hone et al., supra) of the targetstrain.

Gene sequences can be made synthetically using, for example, an AppliedBiosystems ABI™ 3900 High-Throughput DNA Synthesizer (Foster City,Calif. 94404 U.S.A.) using procedures provided by the manufacturer. Tosynthesize large sequences i.e. greater than about 200 bp, a series ofsegments of the full-length sequence are generated by PCR and ligatedtogether to form the full-length sequence using procedures well know inthe art. However, smaller sequences, i.e. those smaller than about 200bp, can be made synthetically in a single round.

Recombinant plasmids may be introduced into bacterial strains byelectroporation using, for example, a BioRad Gene-Pulser. Nucleotidesequencing to verify cDNA sequences may be accomplished by standardautomated sequencing techniques (e.g. using an Applied Biosystemsautomated sequencer, model 373A). DNA primers for DNA sequencing andpolymerase chain reaction (herein referred to as “PCR”) may be producedsynthetically.

In some embodiments of the invention, the bacteria that are geneticallyengineered are attenuated invasive Shigella flexneri and the genes thatare introduced into the bacteria are the adenovirus VAI genes, NSP1 ofrotavirus, and/or NS1 of influenzae virus which are cloned under thecontrol of a eukaryotic promoter and are introduced into the bacteriumby electroporation.

The present invention also provides preparations for administering therecombinant bacterial expression vectors of the invention. Inparticular, vaccine preparations for use in eliciting immune responsesare provided. The preparations include at least one geneticallyengineered bacterial strain as described herein, and a pharmacologicallysuitable carrier. The preparation of such compositions (e.g. for use asvaccines) is well known to those of skill in the art. Typically, suchcompositions are prepared either as liquid solutions or suspensions,however, solid forms such as tablets, pills, powders and the like arealso contemplated. Solid forms suitable for solution in, or suspensionin, liquids prior to administration may also be prepared. Thepreparation may also be emulsified. The active ingredients may be mixedwith excipients that are pharmaceutically acceptable and compatible withthe active ingredients. Suitable excipients are, for example, water,saline, dextrose, raffinose, glycerol, ethanol and the like, orcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like. The vaccine preparations of thepresent invention may further comprise an adjuvant, suitable examples ofwhich include but are not limited to Seppic, Quil A, Alhydrogel, etc.

If it is desired to administer an oral form of the composition, variousthickeners, flavorings, diluents, emulsifiers, dispersing aids orbinders and the like may be added. The composition of the presentinvention may contain any such additional ingredients so as to providethe composition in a form suitable for administration. The final amountof recombinant bacteria in the formulations may vary. However, ingeneral, the amount in the formulations will be from about 1-99 percent.Further, the preparations of the present invention may contain a singletype of recombinant bacteria or more than one type of recombinantbacteria.

In the case of vaccine preparations, the present invention also providesmethods of eliciting an immune response to antigens encoded by thebacterium, and methods of vaccinating a mammal against diseases orconditions associated with such antigens. By eliciting an immuneresponse, we mean that administration of the vaccine preparation of thepresent invention causes the synthesis of specific antibodies (at atiter in the range of 1 to 1×10⁶, preferably 1×10³, more preferable inthe range of about 1×10³ to about 1×10⁶, and most preferably greaterthan 1×10⁶) and/or cellular proliferation, as measured, e.g. by ³Hthymidine incorporation. The methods involve administering a compositioncomprising a bacterial strain of the present invention in apharmacologically acceptable carrier to a mammal. The vaccinepreparations of the present invention may be administered by any of themany suitable means which are well known to those of skill in the art,including but not limited to by injection, orally, intranasally, byingestion of a food product containing the recombinant bacteria, etc. Inpreferred embodiments, the mode of administration is oral, subcutaneous,intradermal or intramuscular.

The invention is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Induction of Type I Interferon Response in Host Cellsby a Recombinant Shigella Vector

The ability of bacteria to induce a type 1 interferon response inmammalian cells was tested and the nature of the response was analyzed.Experimental conditions were as follows: Semi-confluent monolayers ofHeLa cells were exposed to Shigella flexneri carrying a RNA passengermolecule for 1 hour at a multiplicity of infection (MOI) of 100 in a 6well plate at 37° C. Cells were washed twice with Dulbecco's ModifiedEagles's Medium (DMEM). Medium containing 150 μg/ml gentamicin was addedto the cells for 1 hour to kill extracellular bacteria. Subsequently,cells were washed twice, and DMEM with 10% fetal bovine serum (FBS) wasadded and the infected cells were allowed to incubate for 20 h. Cellswere then washed twice with phosphate buffered saline (PBS) and totalRNA was isolated using an RNeasy mini kit (Qiagen). The HumanInterferons and Receptors RT²Profiler™ PCR Array (SuperarrayBiosciences) was utilized to identify up regulation or down regulationof the expression of 84 interferon related genes.

The results are presented in Table 1. As can be seen, invasion of theshigella vector into the human cells led to transcriptional induction oftype I IFNs and IFN stimulated genes such 2′-5′-oligoadenylatesynthetase (2′-5′-OAS). Of the 89 genes that were surveyed, 74 showedmore than a 2-fold increase in transcription.

In addition, further experiments showed that expression of a reportergene from a plasmid DNA passenger molecule delivered by shigella intoIFN-α/β deficient cells was enhanced compared to the cells having anintact IFN system (FIG. 1).

These results clearly suggest that IFN stimulated genes suppress theexpression of genes from passenger molecules delivered to mammaliancells by bacterial vectors.

TABLE 1 Differential IFN associated gene expression: comparison ofshigella-invaded HeLA cells vs non-invaded HeLa cells. Fold GeneInduction ADAR (adenosine deaminase acting on RNA) 3.37 CNTFR (ciliaryneurotrophic factor receptor) 3.54 CRLF2 (cytokine receptor-like factor2) 3.10 CSF2RA (colony stimulating factor 2 receptor) 2.80 CSF3R (colonystimulating factor 3 receptor) 5.44 CXCL10 (chemokine (C-X-C motif)ligand 10) 649.87 EBI3 (Epstein-Barr virus induced gene 3) 4.30 F3Coagulation factor III (thromboplastin, tissue factor) 2.90 IL20RB(interleukin 20 receptor beta) 1.35 ISG15 (interferon stimulated gene15) 13.87 IFI6 (interferon, alpha-inducible protein 6) 18.69 IFI16(interferon, gamma-inducible protein 16) 5.11 IFI27 (interferon,alpha-inducible protein 27) 52.13 IFI30 (interferon, gamma-inducibleprotein 30) 1.56 IFI35 (interferon-induced protein 35) 4.54 IFI44(interferon-induced protein 44) 5.22 IFI44L (interferon-induced protein44-like) 7.33 IFIH1 (interferon induced with helicase C domain 1) 65.53IFIT1 (interferon-induced protein with tetratricopeptide 12.94repeats-1) IFIT1L (interferon-induced protein with tetratricopeptide13.21 repeats-1-like) IFIT2 (interferon-induced protein withtetratricopeptide 6.47 repeats-2) IFIT3 (interferon-induced protein withtetratricopeptide 19.08 repeats-3) IFITM1 (interferon inducedtransmembrane protein 1) 3.47 IFITM2 (interferon induced transmembraneprotein 2) 0.85 IFNA1 (interferon, alpha 1) 2.34 IFNA14 (interferon,alpha 14) 3.02 IFNA2 (interferon, alpha 2) 19.48 IFNA21 (interferon,alpha 21) 14.66 IFNA4 (interferon, alpha 4) 7.86 IFNA5 (interferon,alpha 5) 37.90 IFNA6 (interferon, alpha 6) 3.28 IFNA8 (interferon, alpha8) 3.77 IFNAR1 (interferon (alpha, beta and omega) receptor 1) 2.44IFNAR2 (interferon (alpha, beta and omega) receptor 2) 3.72 IFNB1(interferon, beta 1) 21.92 IFNE1 (interferon epsilon 1) 1.72 IFNG(interferon, gamma) 6.21 IFNGR1 (interferon-gamma receptor 1) 8.08IFNGR2 (interferon-gamma receptor 2) 3.13 IFNK (interferon, kappa) 5.40IFNW1 (interferon, omega 1) 18.18 IFRD1 (interferon-relateddevelopmental regulator 1) 8.36 IFRD2 (interferon-related developmentalregulator 2) 1.07 IL10RA (interleukin 10 receptor, alpha) 9.67 IL10RB(interleukin 10 receptor, beta) 3.28 IL11RA (interleukin 11 receptor,alpha) 2.02 IL12B (interleukin 12, beta) 31.00 IL13RA1 (interleukin 13receptor, alpha-1) 1.64 IL15 (interleukin 15) 2.59 IL20RA (interleukin20 receptor, alpha) 2.82 IL21R (interleukin 21 receptor) 6.21 IL22RA2(interleukin 22 receptor, alpha-2) 8.78 IL28A (interleukin 28, alpha)5.26 IL28RA (interleukin 28 receptor, alpha) 1.94 IL29 (interleukin 29)25.71 IL2RB (interleukin 2 receptor, beta) 9.47 IL2RG (interleukin 2receptor, gamma) 26.61 IL31RA (interleukin 31 receptor, alpha) 5.22IL3RA (interleukin 3 receptor, alpha) 12.85 IL4R (interleukin 4receptor) 4.33 IL5RA (interleukin 5 receptor, alpha) 3.24 IL6(interleukin 6) 42.34 IL6R (interleukin 6 receptor) 11.91 IL7R(interleukin 7 receptor) 22.38 IL9R (interleukin 9 receptor) 1.91 IRF1(interferon regulatory factor 1) 20.03 IRF2 (interferon regulatoryfactor 2) 3.85 IRF2BP1 (interferon regulatory factor 2 bindingprotein 1) 2.32 IRF2BP2 (interferon regulatory factor 2 binding protein2) 4.94 IRF3 (interferon regulatory factor 3) 1.88 IRF4 (interferonregulatory factor 4) 49.32 IRF5 (interferon regulatory factor 5) 5.75IRF6 (interferon regulatory factor 6) 5.67 IRF7 (interferon regulatoryfactor 7) 3.02 IRF8 (interferon regulatory factor 8) 30.36 IRGM(immunity-related GTPase family, M) 350.68 LEPR (leptin receptor) 2.23MPL (myeloproliferative leukemia protein) 4.64 MX1 (Myxovirus(influenza) resistance 1) 13.40 OAS1 (2′-5′-oligoadenylate synthetase)8.66 PSME1 (proteasome (prosome, macropain) activator sub- 1.13 unit 1)PYHIN1 (pyrin and HIN domain) 2.63 SP110 (nuclear body protein) 1.82 TTN(encodes central sarcomeric protein, titin) 45.07 B2M(beta-2-microglobulin) 2.21 HPRT1 (hypoxanthinephosphoribosyltransferase 1) 0.68 RPL13A (ribosomal protein L13a) 0.49GAPDH (glyceraldehyde-3-phosphate dehydrogenase) 0.98 ACTB (actin, beta)1.39

Example 2 Construction of Bacterial Delivery Systems that Counter theNegative Effects Of the Type I IFN Response on Expression of PassengerNucleic Acids Delivered by Bacterial Vectors

This example describes the construction and use of two bacterialdelivery systems that reduced the negative effects of IFNs on expressionof a passenger nucleic acids. In both cases, nucleic acids weregenetically engineered into attenuated, invasive Shigella flexneristrains by electroporation. Shigella flexneri was selected because it isnaturally invasive in many tissue culture cell lines and animal models.The Shigella strain carries introduced chromosomal mutations that causeit to lyse after invasion of eukaryotic cells and escape from theendocytic vesicle, enabling the release of passenger molecules into theeukaryotic cell cytoplasm.

In the first set of experiments, electro-competent Shigella flexneristrain NCD1 was prepared and electroporated with the commerciallyavailable E. coli beta-galactosidase-expressing reporter vectorpcDNA3.1/His/lacZ (Invitrogen). Reporter vector pcDNA3.1/His/lacZexpresses E. coli beta-galactosidase under the control of the humancytomegalovirus (CMV) promoter in mammalian cells, permitting the readyanalysis of mammalian-mediated gene expression after delivery of thevector. The interferon resistance gene used in this experiment was theadenovirus-associated I (VAI) RNA gene. The adenovirus RNA gene is knownto be transcribed by RNA polymerase III in large amounts afteradenovirus infection (Reich et al., J. Mol. Biol. 17, 428, 1966; Priceet al., J. Virol. 9, 62, 1972; Weinmann et al., Proc. Nat. Acad. Sci.USA 71, 3426; Soderlund et al., Cell 7, 585, 1976.) Adenoviruses use thevirus-encoded virus-associated RNA as a defense against cellularantiviral responses by blocking the activation of theinterferon-induced, double-stranded RNA-activated protein kinase PKR(Galabru J, Katze M G, Robert N, Hovanessian A G. Eur J. Biochem. 1989Jan. 2; 178(3):581-9). The pAdVAntage vector that contains theAdenovirus Virus-Associated I (VAI) RNA gene on a 1,724 bp insert wasalso electroporated into the Shigella flexneri NCD1 strain. Invasion ofHeLa cells by electroporation with Shigella flexneri strains was carriedout as described in Example 1. Briefly, to test the anti-interferoneffect of the VAI gene, HeLa cells were co-invaded with Shigellaflexneri NCD1 containing the beta-galactosidase reporter vector(pcDNA3.1/His/lacZ) and Shigella flexneri NCD1 containing the pAdVAntagevector, or with Shigella flexneri NCD1 strain alone (without a vector).After 24 hours, beta-galactosidase assay reagents (Stratagene) were usedboth for cell lysis and for the assay of beta-galactosidase activity incell extracts. The results are presented in FIG. 2. As can be seen, alarge increase in beta-galactosidase activity was observed in the HeLacells invaded by shigella that contained both the beta-galactosidasereporter vector and the anti-interferon pAdVAntage vector.

Similarly, in the second set of experiments, a shigella vector straincontaining recombinant double-stranded RNA nucleocapsids (rdsRN)carrying the reporter gene Green Fluorescent Protein (GFP) wereelectroporated with sequences encoding influenza-A NS1 or rotavirus NSP1which were cloned into the eukaryotic expression vector pcDNA 3.1 zeo(+)(Invitrogen). The resulting Shigella strain thus contained both the GFPgene in the RNA nucleocapsid (rdsRN) and NSP1 or NS1 in pcDNA. BHK-21and HeLa cells were invaded with the GFP and NSP1 or NS1-expressionplasmid harboring Shigella strain. After 16 hours, invaded HeLa cellswere tested for green fluorescence and the HeLa cell lysate was analyzedfor GFP protein by immunoblotting. The fluorescence results showed thatexpression of GFP protein was enhanced in the cells which were invadedwith an NS1- or NSP1-expression plasmid harboring Shigella strain,compared to cells invaded by a Shigella strain with only a GFP gene(data not shown). Immunoblotting of total protein produced by theeukaryotic cells confirmed higher GFP expression in cells invaded with aNS1 or NSP1-expression plasmid harboring Shigella strain (FIG. 3).

These findings show that expression of a gene encoding an inhibitor ofthe type I interferon response enhances the co-expression of a transgeneencoding a protein of interest (e.g. beta-galactosidase or GFP) enhancesthe expression of the protein of interest. The results described in thisExample are the first evidence showing that enhanced expression of aprotein of interest can be obtained by attenuating the IFN responseusing a bacterial based delivery system.

The same or similar results are obtained in vivo in mammalian cells ortissues invaded by a genetically engineered bacterium encoding anantigen such as a tuberculosis antigen, and a factor that attenuates theIFN response. Expression of the antigen is greater in such a bacteriumthan if the factor was not produced. As a result, the mammalian hostwould successfully mount an immune response to the antigen.

Example 3 Construction of an Expression Vector Expressing an InterferonResistance Gene in Both Bacteria and Mammalian Cells and a Protein ofInterest Only in Mammalian Cells

A plasmid vector is constructed to express the immunodominant gagpeptide of HIV-1. A 600 bp fragment is PCR-amplified from a syntheticgag gene. The sequence is amplified using Accuprime DNA polymerase(Invitrogen, Carlsbad, Calif.) and primers including HpaI and NotI REsites. The size of the amplified sequence is verified by agarose gelelectrophoresis, and is purified using a QIAquick PCR purification kitby following manufacturer's instructions (Qiagen, Cat. No. 28106,Valencia, Calif.). The 600 bp gag gene is cloned into the EcoRV and NotIsites (New England Biolabs, Beverly, Mass.) of the expression vectorplasmid pcDNA3.1zeo(+) (Invitrogen, Carlsbad, Calif.). Recombinantplasmids harboring the appropriate inserts are identified and the novelplasmid is designated pGAG4X.

An interferon resistance gene (e.g. NS1 or NSP1) is cloned into thepGAG4X vector under the control of an appropriate eukaryotic promoter(e.g.SV40 promoter) or prokaryotic promoter (e.g. house keeping promoterof arg1), or both, generating a dual expression vector. (The particulareukaryotic and prokaryotic promoter sequences described herein are notcritical to the construction of the vector and other suitable promoterswill occur to those of skill in the art.) Thus, this expression vectorexpresses an interferon resistance gene in both bacteria and mammaliancells; however the protein of interest (e.g. Gag of HIV-1) is expressedonly in mammalian cells. This approach improves transcript stability ansubsequent translation of passenger RNA/DNA and other molecules for theexpression of foreign proteins of interest or inhibitory RNAs inmammalian cells.

Example 4 Use of a Recombinant Bacterial Expression Vector that isGenetically Engineered to Suppress the IFN Response as a Vaccine

The efficacy of any bacterial live-vector vaccine rests with its abilityto present sufficient foreign antigen to the human immune system toinitiate the desired protective immune response. However, passengerDNA/RNA molecules may become unstable in vivo due to the host defensesystem, namely the IFN response, resulting in the loss of foreign genesand a decrease in the intended immune response. This invention providesa solution for the synthesis of high levels of antigen within host cellsby attenuating the IFN defense system.

Delivery and expression of genes encoding IFN resistance and an antigenof interest may be accomplished by the inoculation of targeted cells(tissue, organism, etc.) with a non-pathogenic or attenuated bacterialvaccine vector that carries nucleic acids encoding the transgene ofinterest and a suppressor of the type I IFN response. Biologicalresponses of interest include, but are not limited to: protective ormodulatory immune responses; therapeutic responses; and down-regulationof gene expression (e.g. siRNA) and up-regulation of gene expression(e.g. cytokine expression) of host proteins.

Once a non-pathogenic or attenuated bacterial vaccine vector strain hasbeen selected, the strain is modified to serve as an interferon responsesuppressing strain. This is accomplished using the strategies describedabove that entail introducing one or more IFN resistance genes into thestrain.

To generate strains that contain type I IFN resistance genes andantigens of interest, in vitro synthesized gene(s) are introduced intothe strains by electroporation and transformants are isolated on solidmedia under conditions that only permit the growth of strains thatharbor and express a positive selection allele in the recombinantplasmid (e.g. antibiotic resistance or complementation of auxotrophy).One method of enhancing the inheritance of expression plasmids by livevectors involves construction of a passenger nucleic acids designed tocomplement an introduced mutation in the bacterial chromosome. In aplasmid-based complementation system, plasmids replicating in thecytoplasm of the bacterium express a critical protein required by thebacterium to grow and replicate; loss of such plasmids removes theability of the bacterium to express the critical protein and results incell death. (The phenomenon of plasmid loss during bacterialreplication, which results in the death of any plasmid-less bacterium,is also referred to as “post segregational killing.”) Such a system hasbeen successfully employed in Salmonella typhimurium and is based onexpression of the asd gene encoding aspartate β-semialdehydedehydrogenase (Asd) (Galen et al., Gene. 1990; 49:29-35). Asd is acritical enzyme involved in the synthesis of structural componentsessential for the formation of the cell wall in gram-negative bacteria.Therefore, loss of plasmids encoding such a critical enzyme would belethal for any bacterium incapable of synthesizing Asd from thechromosome.

The amount of such recombinant bacteria to be administered variesdepending on the species of the subject, as well as the disease orcondition that is being treated. Generally, the dosage employed is about10³ to 10¹¹ viable organisms, preferably about 10³ to 10⁹ viableorganisms. The bacterial vector harboring the DNA/RNA passenger moleculeis generally administered along with a pharmaceutically acceptablecarrier or diluent. The particular phaimaceutically acceptable carrieror diluent employed is not critical to the present invention. Examplesof diluents include a phosphate buffered saline, buffer for bufferingagainst gastric acid in the stomach, such as citrate buffer (pH 7.0)containing sucrose, bicarbonate buffer (pH 10) alone (Levine et al., J.Clin. Invest., 79:888-902; 1987); (Black et al., J. Infect. Dis.,155:1260-1265; 1987), or bicarbonate buffer (pH 7.0) containing ascorbicacid, lactose, and optionally aspartame (Levine et al., Lancet, II: 467470; 1988). Examples of carriers include proteins, e.g., as found inskim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typicallythese carriers would be used at a concentration of about 0.1-90% (w/v)but preferably at a range of 1-10% (w/v).

The biological activity of vector strains is assessed in an appropriateanimal model (e.g. mice, rabbits, guinea pigs or Rhesus macaques).Initially, the bacterial vector strains are administered at doses of10²-10⁹ cfu, and are administered by an appropriate route (e.g. E. coli,Salmonella and Shigella can be given intragastrically or intranasally).The number of doses will vary, depending on the potency of theindividual vector strain, and the valency of the encoded recombinantproduct of interest.

Methods of measurement of immune and other biological responses toencoded products in animal models are well known to those skilled in theart. To measure serum IgG and IgA responses to antigen, sera arecollected before and 10, 20, 30, 40, 50, 60, 70, and 80 days aftervaccination. About 400-500 μl of blood is collected into individualtubes and allowed to clot by incubating for 4 hr on ice. Aftercentrifugation in a microfuge for five minutes, the sera are transferredto fresh tubes and stored at −80° C. Mucosal IgG and IgA responses toantigens expressed by the genes of interest are determined using fecalpellets and vaginal washes that will be harvested before and at regularintervals after vaccination (Srinivasan et al., Biol. Reprod. 53: 462;1995); (Staats et al., J. Immunol. 157: 462; 1996). Standard ELISAs areused to quantitate the IgG and IgA responses to an antigen of interestin the sera and mucosal samples (Abacioglu et al., AIDS Res. Hum.Retrovir. 10: 371; 1994); (Pincus et al., AIDS Res. Hum. Retrovir. 12:1041; 1996). Ovalbumin can be included in each ELISA as a negativecontrol antigen. In addition, each ELISA can include a positive controlserum, fecal pellet or vaginal wash sample, as appropriate. The positivecontrol samples are harvested from animals vaccinated intranasally with10 μg of the antigen expressed by the gene of interest mixed with 10 μgcholera toxin, as described (Yamamoto et al., Proc. Natl. Acad. Sci. 94:5267; 1997). The end-point titers are calculated by taking the inverseof the last serum dilution that produced an increase in the absorbanceat 490 nm that is greater than the mean of the negative control row plusthree standard error values.

Cellular immunity may be measured by intracellular cytokine staining(also referred to as intracellular cytokine cytometry) or by ELISPOT(Letsch A. et al., Methods 31:143-49; 2003). Both methods allow thequantitation of antigen-specific immune responses, although ICS alsoadds the simultaneous capacity to phenotypically characterizeantigen-specific CD4+ and CD8+ T-cells. Such assays can assess thenumbers of antigen-specific T cells that secrete IL-2, IL-4, IL-5, IL-6,IL-10 and IFN- (Wu et al., AIDS Res. Hum. Retrovir. 13: 1187; 1997).ELISPOT assays are conducted using commercially-available capture anddetection mAbs (R&D Systems and Pharmingen), as described (Wu et al.,Infect. Immun. 63:4933; 1995) and used previously (Xu-Amano et al., J.Exp. Med. 178:1309; 1993); (Okahashi et al., Infect. Immun. 64:1516;1996). Each assay includes mitogen (Con A) and ovalbumin controls. Theanti-IFN bacterial based delivery system described herein has severaladvantages over delivery systems without IFN resistant genes. Theantigen genes are expressed at higher levels and for longer periods oftime, and therefore induce a more vigorous immune response. Bacterialvectors that display efficacy and are non-toxic in animal models arefurther assessed in clinical trials.

Example 5 Development of a Tuberculosis Vaccine

BCG bacteria are genetically engineered as described herein to containnucleic acids encoding 1) one or more tuberculosis antigens as passengergenes, and 2) one or more factors that inhibit or interfere with amammalian host cell type I interferon response. When administered to amammalian host (e.g. a human), the genetically engineered BCG invadehost cells, escape the endosome, and are lysed to release passengergenes to produce the one or more tuberculosis antigens. Further, the BCGalso produce the one or more factors that inhibit the host cells IFNresponse. The factors attenuate the host cell IFN response, which wouldotherwise decrease the production of the TB antigen(s). As a result,sufficient TB antigen(s) is produced to result in a robust immuneresponse to the TB antigen(s).

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

We claim:
 1. A method of inducing an immune response to one or moreantigens of interest in a mammal, comprising: administering to themammal a genetically engineered bacterium, comprising: nucleic acidsequences encoding the one or more antigens of interest; and nucleicacid sequences encoding one or more factors that inhibit a mammaliantype I interferon response; wherein the nucleic acid sequences encodingthe one or more antigens of interest are operably linked to a eukaryoticpromoter, and the nucleic acid sequences encoding the one or morefactors that inhibit a mammalian type I interferon response are operablylinked to a eukaryotic promoter or a prokaryotic promoter, and whereinthe step of administering is carried out under conditions which allowthe genetically engineered bacterium to invade a cell or tissue in themammal, and which allow the cell or tissue to produce an immune responseto the one or more antigens of interest.
 2. The method of claim 1,wherein at least one of the antigens of interest is a Mycobacteriumtuberculosis antigen.
 3. The method of claim 1, wherein the nucleic acidsequences encoding the one or more factors that inhibit a mammalian typeI interferon response are operably linked to a eukaryotic promoter. 4.The method of claim 1, wherein the nucleic acid sequences encoding theone or more factors that inhibit a mammalian type I interferon responseare operably linked to a prokaryotic promoter.
 5. The method of claim 1,wherein the nucleic acid sequences encoding the one or more factors thatinhibit a mammalian type I interferon response are present on achromosome of the genetically engineered bacterium.
 6. The method ofclaim 1, wherein one or both of: i) the nucleic acid sequences encodingthe one or more antigens of interest, and ii) the nucleic acid sequencesencoding the one or more factors that inhibit a mammalian type Iinterferon response, are present on a plasmid.
 7. The method of claim 1,wherein the one or more factors that inhibit a mammalian type Iinterferon response are of viral origin.
 8. The method of claim 1,wherein the genetically engineered bacterium is a bacterium selectedfrom the group consisting of Shigella, Listeria, Salmonella, andBacille-Calmette-Guerin (BCG).
 9. The method of claim 1, wherein the oneor more factors that inhibit a mammalian type I interferon response arerotavirus NSP 1 or influenza virus NS1.
 10. A method for inducing animmune response to one or more antigens of interest in a mammal,comprising: administering to the mammal a genetically engineeredbacterium, comprising: one or more genetically engineered nucleic acidsequences coding for a host cell or tissue type 1 interferon (IFN)response suppressor factor; and one or more genetically engineerednucleic acids coding for one or more antigens to which an immuneresponse is desired; wherein said one or more genetically engineerednucleic acids coding for said one or more antigens to which an immuneresponse is desired are over expressed upon said bacterium invading saidhost cell or tissue.
 11. The method of claim 10, wherein the host cellor tissue type 1 IFN response suppressor factor is rotavirus NSP 1 orinfluenza virus NS1.
 12. The method of claim 10, wherein the one or moreantigens to which an immune response is desired are selected fromtuberculosis antigens and malaria antigens.
 13. The method of claim 10,wherein the one or more antigens to which an immune response is desiredis one or more viral antigens.
 14. The method of claim 10, wherein theone or more antigens to which an immune response is desired are selectedfrom hormone, enzymes, anticancer agents, and apoptotic factors.
 15. Themethod of claim 10, wherein the host cell or tissue type 1 IFN responsesuppressor factor is selected from the group consisting of rotavirusNSP1, influenza virus NS1, ectromelia virus C12R protein, hepatitis Cvirus NS3/4A protease, vaccinia virus vIFN-α/β Rc protein, adenovirusE1A protein, C proteins of paramyxoviruses, and human papillomavirus(HPV) E6 oncoprotein.
 16. The method of claim 13, wherein the one ormore viral antigens is a rotavirus viral antigen, influenza virus viralantigen, ectromelia virus viral antigen, hepatitis virus viral antigen,vaccinia virus viral antigen, adenovirus viral antigen, paramyxovirusviral antigen, HPV viral antigen, HIV viral antigen, HTLV viral antigen,enterovirus viral antigen, herpesvirus viral antigen, EEE viral antigen,VEE viral antigen, West Nile virus viral antigen, Norwalk virus viralantigen, parvovirus viral antigen, dengue virus, viral antigen orhemorrhagic fever virus viral antigen.